As a result of genomic analyses, an attempt has been actively made to apply genomic information regarding genetic polymorphisms and the like to the medical field.
For example, it is said that single nucleotide polymorphisms (SNPs) exist in 1 out of 1,000 nucleotides. The SNPs are considered to cause individual differences and differences in individual characteristics or congenital predispositions. Moreover, it has being revealed that factor genes are associated, as risk factors, with diseases (diabetes, hypertension, etc.) on which environmental factors had previously been considered to act at a relatively high rate. Also, it has being revealed that many such factor genes relate to single nucleotide polymorphisms. Hence, SNPs analysis has been considered to result in medication and treatment that are adapted to the physical constitutions of individuals (Taylor-made medical treatment), and thus the SNPs analysis has got a lot of attention.
The subtypes of bacteria or viruses, such as HCV, influenza virus or Helicobacter pylori, have been considered to be certain types of polymorphisms. The therapeutic effects of various types of agents are different depending on such subtypes. Accordingly, the analyses of the polymorphisms of such viruses and the like would give information important for selection of treatment methods.
As a method for discriminating between the nucleotide sequences of nucleic acids, there has been known a method for carrying out a nucleic acid amplification reaction using DNA polymerase and primers designed to have sequences complementary to sequences to be detected.
As a method of amplifying a nucleic acid, a polymerase chain reaction (PCR) has been widely known. In order to amplify a target nucleic acid sequence of interest, the PCR method comprises the following three steps: a step of denaturing double-stranded DNA used as a template to convert it to single-stranded DNA (denaturation step); a step of annealing a primer to the single-stranded DNA (annealing step); and elongating a complementary stand using the primer as an origin (elongation step). In an ordinary PCR method, a thermal cycler is used, and the denaturation step, the annealing step and the elongation step are carried out at different temperatures. However, in order to carry out a nucleic acid amplification reaction at the 3 different types of temperatures, it is necessary to precisely control temperatures, and thereby it becomes difficult to carry out tests simply using small devices. Moreover, the PCR method has also been problematic in that time loss increases as the number of the cycle increases.
Under the aforementioned circumstances, a nucleic acid amplification method that can be carried out under isothermal conditions has been developed. Examples of such a nucleic acid amplification method include RCA (Rolling Circle Amplification: Proc. Natl. Acad. Sci, Vol. 92, 4641-4645 (1995)), an SDA method (Strand Displacement Amplification; JP Patent Publication (Kokai) No. 5-130870 A (1993)), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids), LAMP (Loop-Mediated Isothermal Amplification of DNA; Bio Industry, Vol. 18, No. 2 (2001)), NASBA (Nucleic acid Sequence-based Amplification method; Nature, 350, 91-(1991)), and TMA (Transcription mediated amplification method; J. Clin Microbiol. Vol. 31, 3270-(1993)).
However, these methods have been problematic in that they cause high costs for the combined use of exonuclease, RNaseH, reverse transcriptase or the like with polymerase, or for the use of specific primers, and also in that it is extremely difficult to design primers.
JP Patent Publication (Kokai) No. 2002-233379 describes a method for amplifying the DNA of a region of interest by performing a reaction at an isothermal temperature using at least a pair of oligonucleotide primers in the presence of DNA polymerase having strand displacement ability. However, the method described in JP Patent Publication (Kokai) No. 2002-233379 has been problematic in that it requires a relatively long reaction time.
Furthermore, methods for discriminating between the nucleotide sequences of nucleic acids using a nucleic acid amplification reaction share a problem that such amplification reaction often takes place even when primers are not completely complementary to a target sequence (non-specific amplification). For example, in the case of discriminating between single nucleotide polymorphisms, a difference between a nucleic acid as a target to be discriminated and a nucleic acid as a non-target to be discriminated is only one nucleotide. As a result, the aforementioned non-specific amplification significantly takes place. Whether or not such non-specific amplification takes place depends also on subtle conditions such as apparatuses or surrounding environments. Accordingly, it is difficult to suppress such non-specific amplification.